Amphiphilic cationic poly(ethylene oxide)-S(polylysine)-poly(d,l-lactide) (mPEO-S(CKn)-PLA) tri-arm star copolymers were synthesized by a combination of ring opening polymerization (ROP) and a thiol-disulfide exchange. The mPEO-S(CKn)-PLA copolymers were found to be non-cytotoxic and could effectively condense GFP plasmid DNA into nanometer-sized complexes, as characterized by dynamic light scattering (DLS), suitable for endocytotic cellular uptake. In vitro DNA transfection studies showed that the amphiphilic structure is capable of DNA transfection and GFP expression. Addition of chloroquine into the medium further enhanced the DNA transfection efficiency. (c) 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018, 56, 635-644